Abstract
Cytoplasmic dynein powers intracellular movement of cargo toward the microtubule minus end. The first step in a variety of dynein transport events is the targeting of dynein to the dynamic microtubule plus end, but the molecular mechanism underlying this spatial regulation is not understood. Here, we reconstitute dynein plus-end transport using purified proteins from S. cerevisiae and dissect the mechanism using single-molecule microscopy. We find that two proteins-homologs of Lis1 and Clip170-are sufficient to couple dynein to Kip2, a plus-end-directed kinesin. Dynein is transported to the plus end by Kip2, but is not a passive passenger, resisting its own plus-end-directed motion. Two microtubule-associated proteins, homologs of Clip170 and EB1, act as processivity factors for Kip2, helping it overcome dynein's intrinsic minus-end-directed motility. This reveals how a minimal system of proteins transports a molecular motor to the start of its track.DOI: http://dx.doi.org/10.7554/eLife.02641.001.
Highlights
Cytoskeletal motor proteins transport and position a variety of macromolecules, organelles and mRNAs in the cell interior (Vale, 2003), but how these motors are themselves targeted to specific locations within the cell is an important unsolved question
Live-cell imaging in diverse organisms reveals that, surprisingly for a minus-end-directed motor, dynein accumulates at the plus ends of microtubules that grow and shrink near the cell periphery (Vaughan et al, 1999; Han et al, 2001; Ma and Chisholm, 2002; Lee et al, 2003; Sheeman et al, 2003; Lenz et al, 2006; Kobayashi and Murayama, 2009)
We began by purifying the S. cerevisiae proteins Lis1, Bik1, Bim1 and Kip2, in addition to a wellcharacterized dynein motor construct (GST-dynein331 kDa; referred to as ‘dynein’) (Reck-Peterson et al, 2006)
Summary
Cytoskeletal motor proteins transport and position a variety of macromolecules, organelles and mRNAs in the cell interior (Vale, 2003), but how these motors are themselves targeted to specific locations within the cell is an important unsolved question. Cytoplasmic dynein, a large and complex AAA+ motor protein (Carter, 2013), uses the energy from ATP hydrolysis to move cargoes toward the minus end of microtubules (typically toward the cell center). Live-cell imaging in diverse organisms reveals that, surprisingly for a minus-end-directed motor, dynein accumulates at the plus ends of microtubules that grow and shrink near the cell periphery (Vaughan et al, 1999; Han et al, 2001; Ma and Chisholm, 2002; Lee et al, 2003; Sheeman et al, 2003; Lenz et al, 2006; Kobayashi and Murayama, 2009). By localizing to dynamic microtubule plus ends (Howard and Hyman, 2009), dynein is thought to ‘search-and-capture’ (Kirschner and Mitchison, 1986) cargo molecules, before transporting them toward the minus end (Wu et al, 2006). The molecular mechanisms that target dynein to the microtubule plus end are poorly understood
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